This project employs a range of structural and biochemical techniques to examine normal synaptic plasticity and development. These approaches have in common their dependence on rapid~freezing and direct visualization of living brain by light microscopic techniques. This project has been engaged in exploring various live brain preparations suitable for these purposes. Recently, success has been achieved with a new approach to culturing hippocampal slices. Their typical laminar organization and most of their thickness can be maintained for up to 12 weeks by culturing them at the interface between air and culture medium. The slices also maintain their normal electrophysiological function. Now, ultra-structural studies of the slices are being carried out on cultures of different ages using both chemically fixed and freeze-substituted preparations. Preliminary observations indicate modifications of synaptic morphology in long-term cultured slices. After five weeks in culture, many concave~shaped spines appeared, and some had "spinules", finger-like indentations of the postsynaptic membrane projecting into the presynaptic terminal. After nine weeks in culture, the number of concave-shaped spines and spinule-containing spines decreased, but the presynaptic terminal enlarged and many segmented synaptic active zones appeared. These structural changes are reminiscent of previously reported activity-dependent changes in synaptic morphology. Taken together, these results show that hippocampal organotypic cultures, prepared according to our new method, will be an excellent preparation to examine synaptic plasticity and development.